Field of the Invention
The invention relates to multi-photon excitation microscopy, and in particular to a planner and calibration module for a multi-mirror adaptive sampling system configured to maintain a high frame rate during wide field of view sampling.
Background of the Invention
In laser scanning microscopy, a laser is directed at a sample such as animal tissue and an image is acquired one pixel at a time. Two-photon excitation microscopy is a fluorescence imaging technique that allows three-dimensional imaging of living tissue up to about one millimeter in depth. In contrast to confocal laser scanning microscopy, it does not require a focal diaphragm. Being a special variant of the fluorescence microscope, it uses red-shifted excitation light to excite fluorescent dyes causing an emission of photons. However, for each excitation, two photons of infrared light are absorbed. Using infrared light minimizes scattering in the tissue. Due to the multiphoton absorption, the background signal is strongly suppressed. Both effects lead to an increased penetration depth for these microscopes. Two-photon excitation, and now three-photon excitation, can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection, and reduced phototoxicity.